보고서 정보
주관연구기관 |
단국대학교 DanKook University |
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2015-02 |
과제시작연도 |
2014 |
주관부처 |
농촌진흥청 Rural Development Administration(RDA) |
등록번호 |
TRKO201500010723 |
과제고유번호 |
1395035607 |
사업명 |
차세대바이오그린21 |
DB 구축일자 |
2015-07-11
|
DOI |
https://doi.org/10.23000/TRKO201500010723 |
초록
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Ⅳ. 연구개발결과
단자엽 화훼 작물인 글라디올러스의 polyubiqutin 유전자인 GUBQ1 프로모터의 5’-UTR intron을 담배의 polyubiqutin 유전자의 5‘ UTR-intron과 대체하여 토마토 담배 등과 같은 가지과 작물에서 활성이 높은 전신발현 프로모터 개발함 또한 다종 바이러스 저항성 형질전환 백합 개발을 위해 CMV, LMoV, LSV 바이러스에 저항성을 가지는 Vector(pPZP-RNAiLV1-Bar,pPZP-RNAiLV2-Bar, and pPZP-RNAiLV3-Bar)를 성공적으로 제작하였다.
Ⅳ. 연구개발결과
단자엽 화훼 작물인 글라디올러스의 polyubiqutin 유전자인 GUBQ1 프로모터의 5’-UTR intron을 담배의 polyubiqutin 유전자의 5‘ UTR-intron과 대체하여 토마토 담배 등과 같은 가지과 작물에서 활성이 높은 전신발현 프로모터 개발함 또한 다종 바이러스 저항성 형질전환 백합 개발을 위해 CMV, LMoV, LSV 바이러스에 저항성을 가지는 Vector(pPZP-RNAiLV1-Bar,pPZP-RNAiLV2-Bar, and pPZP-RNAiLV3-Bar)를 성공적으로 제작하였다. 이를 이용하여 바이러스 저항성 유전자가 삽입된 형질전환체 약 800개 개체를 확보 하였으며 그중 약 300 형질전환체에서 One copy가 도입되었음을 확인하였으며, 원예적 특성을 규명하고자 하였다.
국내 감염 다종바이러스 저항성 Gm 감자 327개 이벤트를 제작 및 유지하였고, 28종의 분자생물학적 분석(Flanking DNA sequencing을 수행)을 완료 하였고, 그중 5종의 GM 감자에서 single copy를 확인하였으며 진딧물 매게를 xdh한 바이러스 접종 후 저항성 평가를 수행한 결과PVYO, PVA 및 PLRV 에서 뚜렷한 저항성 특성이 확인되었다. 포장재배 결과 바이러스 감염율이 0% 였으며 연차별 감염율 조사가 필요할 것이다.
Abstract
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○ 다종바이러스 저항성 GM 백합 이벤트 개발(PJ009568)
Experiments were conducted to transform lily plants with virus genome sequences that confer virus resistance to the major viruses of lilies, CMV, LMoV, and LSV. T-DNA vectors, pFGC and pPZP-bar. Finally almost 300 plantels were obtained from the cultures transf
○ 다종바이러스 저항성 GM 백합 이벤트 개발(PJ009568)
Experiments were conducted to transform lily plants with virus genome sequences that confer virus resistance to the major viruses of lilies, CMV, LMoV, and LSV. T-DNA vectors, pFGC and pPZP-bar. Finally almost 300 plantels were obtained from the cultures transformed with pPZP-Vector and 254 plantes were obatined from pFGC-Vector. leaflets of 63 transgenic plantles in pPZP vector and 56 transgenic plantlet in pFGC Vector were detached and used for PCR analysis, bar, virus gene were positively amplified only in the transformed confirming the introduction of the trans gene in the lilies. Inverse PCR analysis of 13 transgenic lines, 50% transgenic lilies in pPZP vector and 20% transgenic lilies in pFGC Vector showed one copy. one sequences matched withth known sequences such as Xylanase, which indicated that insertion was occurred within a coding sequence of the genomic lily.
Transgenic plants were assessed for resistance to infection by CMV, LSV, all asnsmitted by a natural vector the cotton plant apid, Aphis gossypii Glover. resistance was assessed by the absence of detectable virus accumulation in the foliage. using the rt PCR assay, Both transgenic plants and non transgenic plants determining infected virus CMV, LSV from aphids. but can't compared whether transgenic plant have resistant by virus. plant characteristics such as bulb weight, average bulblet diameter, number of leaves, leaf length were no difference with non transgenic lily cultivar.
○ 화훼류 이벤트 개발 지원 및 프로모터 개발 (PJ00956803)
- Polyubiquitin gene promoter was isolated from Gladiolus (GUBQ1) and this promoter showed high level and constitutive expression.
- Plant expression vectors were constructed using 5'-UTR introns, which were isolated from tomato and tobacco. These 5'-UTR introns were substituted for Gladiolus 5'-UTR intron in GUB Q1 promoter. Plant expression vector of GUBQ1 with tobacco derived 5'-UTR (pBI121::G1-3 Toba) showed higher activity than GUBQ1 in tomato, tobacco and arabidopsis
- Vital coat protein, replcase, Hc-Pro genes were isolated from CMV, LMoV and LSV. Using these genes, pPZP-RNAiLV1-Bar, pPZP-RNAiLV2-Bar, and pPZP-RNAiLV3-Bar Si RNA vectors were constructed for development multi virus resistance transgenic lilis.
- Using the qantitative real-time PCR assay, determining transgene copy number in transgenic plants.
○국내 재배용 다종 바이러스 저항성 GM감자 개발 (PJ00956802)
Transgenic potato plants of Solanum tuberosum cultivar Sumi were generated that expressed fused, tandem, 200 bp segments derived from the capsid protein coding sequences of potato virus Y (PVY strain O) and potato leafroll virus (PLRV), as well as the cylindrical inclusion body coding sequences of potato virus A (PVA), as inverted repeat double-stranded RNAs, separated by an intron. The orientation of the expressed double-stranded RNAs was either sense-intron-antisense or antisense-intron-sense RNAs, and the double-stranded RNAs were processed into small RNAs. Four lines of such transgenic potato plants were assessed for resistance to infection by PVY-O, PLRV, or PVA, all transmitted by a natural vector, the green-peach aphid, Myzus persicae. Resistance was assessed by the absence of detectable virus accumulation in the foliage. All four transgenic potato lines tested showed 100% resistance to infection by either PVY-O or PVA, but variable resistance to infection by PLRV, ranging from 72 to 96% in different lines. This was regardless of the orientation of the viral inserts in the construct used to generate the transgenic plants and the gene copy number of the transgene. This demonstrates the potential for using tandem, fused viral segments and the inverted-repeat expression system to achieve multiple virus resistance to viruses transmitted by aphids in potato.
○ 형질전환 감자의 증식 및 농업특성 평가 (ATIS세부과제번호:PJ00956801)
Yield loss owing to virusr infection is the most serious problem in potato production in the field. Even though there are various genetic resources of virus resistance in wild species such as Solanum stoloniferum, but the introgression by back crosses of R genes are time consuming. Therefore transgenic approach with coat protein or RNA silencing is the prominent methodology to breed virus resistance potato. In this present study, we evaluate the resistance of genetically modified potato harboring three different coat protein in one casste vector that show virus resistance to multiple virus, PVY(potato virus Y), PLRV(potato leafroll virus) and PVA(potato virus A) and agricultural characteristics in the field condition. Plant characteristics such as growth habit, foliage structure, flowering time, leaf shape, flower color were no difference with non transgenic potato cultivar, Superior. Tuber characteristics, we analyzed tuber shape, skin color and flesh color and no difference with non transgenic potato cultivar, Superior. But in the yield analysis, among the three lines, one line(600-3-2) was shown low yield compared to control variety. We also analyzed nutritional components of the total protein, vitamin C content, total fat, ash and 17 amino acids in the GM potato and there is no significance difference with non transgenic potato. For the virus resistance, we could not any symptoms in the field, ELISA and PCR test in transgenic and non-tansgenic potato. This results is owing to only first year field trial after acclimatization from in-vitro plant, so we need more field trial to confirm virus resistance of potato in the field trial.
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